CN105647821A - Trichoderma reesei CstrxR1, and building method and application thereof - Google Patents

Trichoderma reesei CstrxR1, and building method and application thereof Download PDF

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CN105647821A
CN105647821A CN201610174130.6A CN201610174130A CN105647821A CN 105647821 A CN105647821 A CN 105647821A CN 201610174130 A CN201610174130 A CN 201610174130A CN 105647821 A CN105647821 A CN 105647821A
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cstrxr1
filamentous fungi
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龙良鲲
丁少军
丁达繁
徐梅娟
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Nanjing Forestry University
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Abstract

The invention discloses a strain of trichoderma reesei CstrxR1, and a building method and application thereof. The trichoderma reesei CstrxR1 has the classification name of trichoderma reesei CstrxR1 and is collected in CCTCC (China Center for Type Culture Collection); the collection date is March 1st, 2016; the collection number is CCTCC NO: M2016078; the collection address is Wuhan, Hubei, China, Wuhan University. The trichoderma reesei CstrxR1 has the advantages that each cellulose yield is improved by 0.78 times through being compared with the trichoderma reesei parent strain. The trichoderma reesei CstrxR1 has important application values on the improvement of the cellulose production efficiency.

Description

One strain Filamentous fungi engineering bacteria CstrxR1 and construction method thereof and application
Technical field
The invention belongs to engineering bacteria technical field, be specifically related to strain Filamentous fungi engineering bacteria CstrxR1 and an application thereof.
Background technology
Cellulase is by the general name of one group of enzyme system that cellulose degradation is glucose monomer, to generally include endo-type glucanase, circumscribed-type glucanase and beta-glucosidase. Cellulase resource is significant for sustainable use biomass resource, can be widely applied to bioenergy, feedstuff, washing, papermaking and Plant source active substances and the field such as prepares. Filamentous fungi (Trichodermareesei) complete cellulase system can be produced, it is the critical strain of industrial cellulase production. Currently, traditional mutation breeding technologies is difficult to continue to lift up the ability of Filamentous fungi cellulase-producing. The cellulase improveing Filamentous fungi by genetic engineering means produces ability, becomes the effective way reducing cellulase production cost.
The cellular redox state of filamentous fungi can control the expression regulation of many important gene. Thioredoxin system (thioredoxinsystem) is present in the antioxidizing system in most organism, has the multiple critical functions such as regulating cell redox equilibrium. Thioredoxin becomes reduction-state to depend on the catalytic action of thioredoxin reductase (thioredoxinreductase, TrxR) from oxidation state. Thus, TrxR has important regulating and controlling effect for the redox state of the redox equilibrium in born of the same parents or some protein molecular. Filamentous fungi cell has high-caliber TrxR and can change its cellular redox state greatly, and then strengthen the gene expression of associated protein (such as cellulase) molecule.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, it is an object of the invention to provide a strain Filamentous fungi engineering bacteria CstrxR1, can High Cellulase Production. It is a further object of the present invention to provide the construction method of above-mentioned Filamentous fungi engineering bacteria CstrxR1.
Technical scheme, in order to realize foregoing invention purpose, the technical solution used in the present invention is:
One strain Filamentous fungi engineering bacteria CstrxR1, specific name is Filamentous fungiTrichodermareeseiCstrxR1, is preserved in China typical culture collection center, and preservation date is: on March 1st, 2016, deposit number is CCTCCNO:M2016078, and preservation address is: Hubei China Wuhan Wuhan University.
In described Filamentous fungi engineering bacteria CstrxR1, clone has the thioredoxin reductase gene order coming from worm plan wax bacterium, and this gene order is such as shown in SEQIDNO.1.
The method building described Filamentous fungi engineering bacteria CstrxR1: the such as DNA fragmentation shown in SEQIDNO.1 imports Filamentous fungi genome by plasmid pAg1-CstrxR1 by sequence, and described plasmid pAg1-CstrxR1 is at plasmid pAg1-H3KpnI andApaI site order loads Filamentous fungi cellobiohydrolase I promoter sequence Pcbh1, the sequence such as DNA fragmentation shown in SEQIDNO.1 and Filamentous fungi cellobiohydrolase I terminator sequence Tcbh1.
Described Filamentous fungi is Filamentous fungi D-86271.
Described Filamentous fungi engineering bacteria CstrxR1 application in cellulase-producing.
Beneficial effect: compared with prior art, the present invention successfully constructs Filamentous fungi engineering bacteria CstrxR1, being experimentally confirmed high expressed worm and intend the Filamentous fungi engineering bacteria of thioredoxin reductase encoding gene in wax bacterium, its yield of cellulase (filter paper enzyme activity) improves 0.78 times compared with parent's Filamentous fungi. Raising cellulase production efficiency in commercial production is had important using value by the present invention.
Accompanying drawing explanation
Fig. 1 is the figure of expression plasmid pAg1-CstrxR1;
Fig. 2 is geneCstrxR1Recombinant protein expression SDS-PAGE electrophoresis detection figure;
Fig. 3 isCstrxR1The qualification figure of gene Filamentous fungi transformant;
Fig. 4 is the comparison diagram of Filamentous fungi engineering strain yield of cellulase under solid state fermentation conditions; D-86271 is unconverted parent strain, and CstrxR1 is Filamentous fungi genetic engineering bacterium, and CK is that the Filamentous fungi containing empty carrier converts bacterial strain;
Fig. 5 is the comparative result figure of Filamentous fungi engineering strain extracellular protein under solid state fermentation conditions; D-86271 is unconverted parent strain, and CstrxR1 is Filamentous fungi genetic engineering bacterium, and CK is that the Filamentous fungi containing empty carrier converts bacterial strain;
Fig. 6 is the comparative result figure of Filamentous fungi engineering strain Biomass under solid state fermentation conditions; D-86271 is unconverted parent strain, and CstrxR1 is Filamentous fungi genetic engineering bacterium;
Fig. 7 is the relative expression quantity result figure of Filamentous fungi engineering strain fermentation culture process cellulase synthetic gene; Wherein, D-86271 is unconverted parent strain, and CstrxR1 is Filamentous fungi genetic engineering bacterium.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further. The experimental technique used in following embodiment if no special instructions, is conventional method. Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
The structure of embodiment 1 expression vector pAg1-CstrxR1
1, worm intends wax bacterium thioredoxin reductase encoding geneCstrxR1Acquisition andE.coliExpress
Analysis worm plan wax bacterium (CeriporiosissubvermisporaB) whole genome sequence (No. GenBank: AEOV00000000.1), it is thus achieved that the thioredoxin reductase protein coding gene of a candidateCstrxR1. Employing Trizol reagent separation worm plan wax bacterium (Ceriporiopsissubvermispora) FP-105752-Sp(is by ForestProductsLaboratory(CentreforForestMycologyResearch), Madison, WI, USA provide) total serum IgE, and with Reverse Transcription box (TransGen, Beijing) carry out synthesis (oligo(dT) primer of cDNA the first chain), and as template with primer CstrxR_f1(containingNdeI recognition sequence and protection base) and CstrxR_r1(containingHindIII recognition sequence and protection base) amplification acquisitionCsTrxRThe cDNA fragment of 1.The cDNA fragment of acquisition is cloned into carrier pEASY-Blunt(TransGen, Beijing), and carried out sequencing analysis (Invitrogen by Shanghai Ying Jun biotech company, Shanghai), correct through sequence verification, its sequence is such as shown in SEQIDNO.1, thus it is speculated that show that the protein sequence of correspondence is such as shown in SEQIDNO.2. The sequence of each primer is as follows:
CstrxR_f1:5 '-gctctagacatatgatggcacccctcacgaatg-3 ';
CstrxR_r1:5 '-cgaagcttatcttcgataccctcttcctc-3 '.
RightCstrxRThe cDNA fragment of 1 gene carries outNdeI/HindIII double digestion, and connect into escherichia coli (E.coli) expression vector pET23b(Novagen, Germany) and corresponding site, it is thus achieved that plasmid pET-CstrxR1. Illustrate according to product operation, pET-CstrxR1 is proceeded to E. coli expression strains (E.coli) Rosseta bacterial strain (TransGen, Beijing), and carry out abduction delivering with isopropyl-1-sulfo-��-D galactopyranose (IPTG). By the CstrxR1 recombiant protein expressed, there is the histidine-tagged of C end fusion, it is carried out His Bind post (Novagen, Germany) purification, and carries out SDS-PAGE electrophoresis detection (as shown in Figure 1). Fig. 1 shows, it is thus achieved that recombinant C strxR1 molecular size and theoretical molecular (38.4kD, including His label) be consistent.
2, Filamentous fungi gene expression plasmid pAg1-CstrxR1 is built
With Filamentous fungi (Trichodermareesei) D-86271(=Rut-C30) (buying in VTTculturecenter, Finland) genomic DNA is template, under high-fidelity enzyme (Fastpfu) acts on, with primer Tcbh1_f(containingXhoI recognition sequence and protection base)/Tcbh1_r(containingApaI recognition sequence and protection base) expand cellobiohydrolase I terminator sequence (Tcbh1) obtaining long 1088bp, and it is inserted into pAg1-H3(Zhang, A.; Lu, P., Dahl-Roshak; A.M., Paress, P.S.; Kennedy, S., Tkacz; J.S.; An, Z., 2003.Efficientdisruptionofapolyketidesynthasegene(pks1) requiredformelaninsynthesisthroughAgrobacterium-mediatedtransformationofGlarealozoyensis.MolGenGenomics268,645-655.)XhoI/ApaI site, it is thus achieved that plasmid pAg1-Tcbh1. Meanwhile, with Filamentous fungi (Trichodermareesei) D-86271 genomic DNA is template, obtains 891bp cellobiohydrolase I promoter sequence (Pcbh1) with primer Pcbh1_f/Pcbh1_r amplification. With plasmid pET-CstrxR1 for template, obtain CstrxR1 gene order with primer CstrxR_f2/CstrxR_r2 amplification. By HieffCloneTMOneStepPcr Cloning Kit (Yi Sheng bio tech ltd, Shanghai, Shanghai), Pcbh1 and CstrxR1 gene order homologous recombination mode achieved above is cloned into plasmid pAg-Tcbh1(to pass throughKpnI/XhoI double digestion), produce plasmid pAg1-CstrxR1. The physical map of gene expression plasmid pAg1-CstrxR1 is as shown in Figure 2. Each primer sequence is as follows:
CstrxR_f2:5 '-catgtctagaatggcacccctcacgaatg-3 ';
CstrxR_r2:5 '-ctttcgcacggagctctcgagctagtggtggtggtggtggtgctc-3 ';
Pcbh1_f:5 '-agtgaattcgagctcggtaccgatagcagtgtctagtagca-3 ';
Pcbh1_r:5 '-tgaggggtgccattctagacatgatgccagtccgcggttg-3 ';
Tcbh1_f:5 '-gactcgagagctccgtgcgaaagcctgacgca-3 ';
Tcbh1_r:5 '-ctgggcccatcgtaaccgagaatccagagctg-3 '.
Embodiment 2 construction expressionCstrxR1The Filamentous fungi engineering strain of gene
1, gene expression plasmid pAg1-CstrxR1 converts Agrobacterium tumefaciems
Take 1 �� g plasmid pAg1-CstrxR1 DNA add Agrobacterium tumefaciems (Agrobacteriumtumefaciens) AGL-1(Mullins, E.D., Chen, X., Romaine, P., Raina, R., Geiser, D.M., Kang, S., 2001.Agrobacterium-mediatedtransformationofFusariumoxysporum:anefficienttoolforinsertionalmutagenesisandgenetransfer.Phytopathology.91:173-180.) competent cell mixes, puts in liquid nitrogen 5 minutes. Take out, add 700 �� LLB fluid mediums 42 DEG C of heat shocks after 2 minutes immediately, 28 DEG C of shaken cultivation 2 hours. Bacterium solution is uniformly coated on the LB agar plate containing 75 �� g/ml kanamycin, is inverted for 28 DEG C and cultivates 2 days, obtain the Agrobacterium tumefaciems list bacterium colony containing recombiant plasmid pAg1-CstrxR1.
The preliminary resistance screening of 2, Agrobacterium tumefaciens mediated genetic transformation and bacterial strain
By obtain containing the Agrobacterium tumefaciems list colony inoculation of recombiant plasmid pAg1-CstrxR1 in 5mLMM fluid medium, 28 DEG C of shaken cultivation 2 days. By IM culture medium, mycelium dilution is arrivedOD 600=0.15,28 DEG C of shaken cultivation 6 hours, extremelyOD 600=0.6. Take 100 �� L bacterium solution and isopyknic Filamentous fungi (Trichodermareesei) D-86271 spore suspension mixing (suspension concentration=2 �� 106Individual spore/mL), it is uniformly coated on CM agar plate (being covered with cellophane), is just putting for 24 DEG C and co-culturing 3 days. It is forwarded on the PDA agar plate containing 75 �� g/mL HYGs and 400 �� g/mL cefalotins by co-culturing thalline, is inverted cultivation for 28 DEG C and grows to the transformant containing hygromycin B resistant gene for 4-5 days.
3, PCR screening is expressedCstrxR1The Filamentous fungi engineering strain of gene
The transformant of the Hygromycin B resistant of acquisition is inoculated in Mandels culture medium (using 1% lactose as carbon source), 28 DEG C, 180rmp shaken cultivation 2 days. Extract its STb gene and RNA according to preceding method after, carry out following PCR and RT-PCR reaction respectively:
PCR(Fig. 3 A): according tohph(hygromycin gene) designs primer hph_f/hph_r, and purpose product is 0.9kb; RT-PCR(Fig. 3 B): according toCstrxRSpecific primer CstrxR_f1/CstrxR_r1, purpose product is 1.032kb; Each primer sequence is as follows:
CstrxR_f1:5 '-gctctagacatatgatggcacccctcacgaatg-3 ';
CstrxR_r1:5 '-cgaagcttatcttcgataccctcttcctc-3 ';
Hph_f:5 '-aagttcgacagcgtctcc-3 ';
Hph_r:5 '-ttccactatcggcgagta-3 '.
Meanwhile, with the Filamentous fungi of unconverted (Trichodermareesei) D-86271, empty carrier pAg1-Tcbh1 Filamentous fungi convert bacterial strain (CK) and sterilized water (NC) for compare, pcr amplification product carries out sepharose electrophoresis detection. Result is as shown in Figure 3. In Fig. 3 A, all can obtain the band of 0.9kb with the bacterial strain of plasmid pAg1-CstrxR1 and the empty carrier pAg1-Tcbh1 Filamentous fungi converted; Unconverted bacterial strain D-86271 and sterilized water (NC) are all without amplified band. In Fig. 3 B, the Filamentous fungi genetic engineering bacterium containing plasmid pAg1-CstrxR1 can obtain the target stripe of 1.032kb, and unconverted bacterial strain D-86271, pAg1-Tcbh1 transformed bacteria (CK) and sterilized water (NC) are all without amplified band.
9 Expression of Plant Heights from screeningCstrxR1Taking a strain in the Filamentous fungi engineering strain of gene and be numbered CstrxR1, on March 1st, 2016 is preserved in China typical culture collection center (CCTCC), specific name be Filamentous fungi (Trichodermareesei) CstrxR1, deposit number is CCTCCNO:M2016078.
Filamentous fungi CstrxR1 bacterium colony under regular culture conditions, in the flocculence of wide paving, is originally the smooth mycelia of white dense, and light green product spore Cong Shu district occurs in back edge, and reverse side is colourless; The short lateral branch of conidiophore mycelia, transparent, multi-branched; Stigma doleiform, middle bent, form substantial amounts of conidium, conidium is oval or elongated, unicellular, transparent, colourless, and wall is smooth, green time in heaps; This bacterium can normally utilize the nitrogenous sources such as the carbon sources such as glucose, starch, glycerol, and peptone, carbamide, ammonium sulfate.
The fermentation of embodiment 3 Filamentous fungi engineering strain and the determination of yield of cellulase
Take Filamentous fungi engineering strain CstrxR1 and be inoculated in PDA inclined-plane, cultivate 7 days, wait conidium to be generated for 28 DEG C. Take 1 �� 108Individual Filamentous fungi spore inoculating is in 100mLMandels culture medium (using 1% glucose as carbon source), and 28 DEG C, 180rpm cultivates 2 days. Take 1mL bacteria suspension to access in solid-state fermentation culture medium, and in 30 DEG C, dark fermentation is cultivated 15 days under 70% damp condition.
Solid-state fermentation culture medium formula: straw (is crushed to 1-3mm and delignification) 1.5g, wheat bran 1.5g, 10 �� Mandels culture medium (with above-mentioned, not carbonaceous sources) 5mL, above component loads in 250-mL triangular flask, fully mixes, autoclaving. The delignification method of straw: add 20mL4%NaOH solution by every 10g straw, after mixing, 121 DEG C, 20min process, and remove brown material with flowing water, the straw after process dries standby.
After fermentation ends, add 30mL sterilized water (containing 0.1%Tween-80) by every triangular flask, stir, and in 28 DEG C, 120rpm vibrates lixiviate 2h. Continue, with the centrifugal 20min of 7000rpm, to be proceeded to by crude enzyme liquid in new centrifuge tube, in 4 DEG C of Refrigerator stores.
The mensuration of filter paper enzyme activity, using WhatmanNO.1 filter paper (3cm �� 0.5cm) as substrate, 50 DEG C of reaction 60min; Endo-type cellulase (EG) live measure using 1% sodium carboxymethyl cellulose (CMC-Na) as substrate, 50 DEG C reaction 30min; Last all with method (Miller, G.L., the 1959.Useofdinitrosalicylicacidreagentfordeterminationofr educingsugar. of 3,5-dinitrosalicylic acid system DNSAnalChem, 31,426-428.) and measure the growing amount of reducing sugar, by standard glucose curve, conversion obtains enzyme and lives (IU/g substrate dry weight). The determination of activity of beta-glucosidase, using 4-nitrobenzophenone-��-D-pyranglucoside (pNPG) of 5mM as substrate, 50 DEG C of reaction 30min, and according to standard curve, calculate enzymatic activity (IU/g substrate dry weight). Above enzyme activity determination all carries out in the citrate buffer solution of the 50mM of pH4.8, often processes 3-4 time and repeats.
Filter paperlyase or CMC enzyme unit enzyme are lived and are defined as: under this experiment condition, and catalytic phase per minute answers substrate to generate the enzyme amount of 1 ��m of ol glucose. Beta-glucosidase enzyme unit enzyme is lived and is defined as: under this experiment condition, and catalysis pNPG per minute generates the enzyme amount of 1 ��m of ol4-nitrophenol.
Result as shown in Figure 4, is expressedCstrxRFilamentous fungi the produced beta-glucosidase of genetic engineering bacterium CstrxR1 of 1 gene, CMC enzyme and Filter paperlyase activity are all remarkably higher than unconverted parent's Filamentous fungi (D-86271). After fermenting 13 days, the yield of the beta-glucosidase of Filamentous fungi genetic engineering bacterium CstrxR1, CMC enzyme and Filter paperlyase adds 0.84,0.48 and 0.78 times than parent strain D-86271 respectively.Meanwhile, the yield of control plasmid pAg1-Tcbh1 transformed bacteria (CK) various enzyme is consistent with unconverted Filamentous fungi parent strain (D-86271).
The analysis of embodiment 4 solid fermentation Filamentous fungi extracellular protein
The fermentation liquid of each bacterial strain in Example 3, by a certain percentage after dilution, measures the extracellular Tot Prot of strain fermentation with BCA detection kit (ThermoTech, USA). Result is as it is shown in figure 5, fermentation 7 to 15 days, the Filamentous fungi genetic engineering bacterium extracellular protein content of CstrxR1 was significantly higher than unconverted Filamentous fungi parent strain D-86271. After fermentation 13 days, the Filamentous fungi genetic engineering bacterium extracellular protein content of CstrxR1 adds 1.4 times than unconverted Filamentous fungi parent strain D-86271.
Embodiment 5, solid ferment process Zhong Ruishi Trichoderma spp. Fungal biodiversity measuring and calculating analysis
By measuring mycelial nucleic acid content in fermentation substrate, indirect method converses the Biomass of thalline. Take 2g solid fermentation thing 70 DEG C, dry 12 hours, and be fully ground in liquid nitrogen to powdery. Take 0.04g abrasive material to put in 1.5mL sterile centrifugation tube, and be sufficiently mixed after adding 1mL5% trichloroacetic acid. Mixture processes 30 minutes (mixing in every 5 minutes is once) in 80 DEG C of water-baths, is placed on 3 minutes on ice. 10,000g are centrifuged 10 minutes, and supernatant proceeds in new centrifuge tube. After diluted sample 100 times, measure the light absorption value under wavelength 260nm, and calculate Fungal biodiversity according to standard curve.
The making of standard curve: cultivate with Mandels culture medium (using 1% glucose as carbon source) and obtain pure Filamentous fungi mycelium, and dry to constant weight. Take 0 respectively, 2.5,5,10,20, the dry mycelia of 40mg measures nucleic acid content therein according to above method, and with dry mycelial weight for vertical coordinate and nucleic acid content for abscissa, draws out standard curve.
Result such as Fig. 6 shows: ferment 5 to 15 days, and the Biomass of Filamentous fungi genetic engineering bacterium CstrxR1 is lower than unconverted Filamentous fungi parent strain D-86271. Therefore,CstrxRThe Fungal biodiversity expressing not increase Filamentous fungi of 1 gene.
The transcription analysis of embodiment 6 Filamentous fungi engineering strain cellulase synthesis related gene
The thalline sample total serum IgE through embodiment 3 fermentation culture 5 days, 7 days, 9 days and 11 Tian Ruishi reesei gene engineered strain CstrxR1 and Filamentous fungi parent strain D-86271 is extracted respectively with Trizol reagent. Take the RNA TransScript RT/RIEnzymeMix(primer oligo(dT of 1 �� g) and 6-mers random primer) (Transgene, Beijing) synthesize cDNA. With this cDNA for template, with primer Qcel1a_f/Qcel1a_r, QeglI_f/QeglI_r and Qcbh1_f/Qcbh1_r respectively to beta-glucosidase synthetic genecel, endo-type cellulase synthetic gene No. 1a(Genbank: XM_006963374)eglAnd cellobiohydrolase synthetic gene No. I(Genbank: XM_006965612)cbhCarry out real-time quantitative PCR (ABIStepOnePlus, USA) No. 1(Genbank: XM_006969162). Response procedures: 94 DEG C of denaturation 30s; 94 DEG C of degeneration 5s, 58 DEG C of annealing 15s, 72 DEG C extend 10s(40 thermal cycle). Negative control without reverse transcription product is set. Meanwhile, with ��-actin gene expression for internal reference, Pfaffl ' s method calculates the relative transcript levels (i.e. relative expression quantity) of target gene, and result is as shown in Figure 7.The sequence of the primer is as follows:
Qcbh1_f:5 '-cttggcaacgagttctctt-3 ';
Qcbh1_r:5 '-tgttggtgggatacttgct-3 ';
Qcel1a_f:5 '-cgtgctcttcaccaacaa-3 ';
Qcel1a_r:5 '-tcttgctgatccacacca-3 ';
QeglI_f:5 '-cttctgctgcaacgagatgg-3 ';
QeglI_r:5 '-tcttggaggtgtcaacggtat-3 ';
Qactin_f:5 '-tccatcatgaagtgcgac-3 ';
Qactin_r:5 '-gtagaaggagcaagagcagtg-3 '.
Wherein primer Qcbh1_f, Qcbh1_r, Qcel1a_f, Qcel1a_r, Qactin_f and Qactin_r is derived from document Xu, J.T., Zhao, G.L., Kou, Y.B., Zhang, W.X., Zhou, Q.X., Chen, G.J., Liu, W.F., 2014.Intracellularbeta-glucosidasesCEL1aandCEL1bareessen tialforcellulaseinductiononlactoseinTrichodermareesei.EukaryoticCell, 13(8): 1001-1013.
Result shows: cellulase synthetic genecel1a��egl1 Hecbh1 expression in Filamentous fungi genetic engineering bacterium (CstrxR1) is significantly higher than unconverted parent Filamentous fungi bacterial strain D-86271, this further demonstrates the cellulase of Filamentous fungi genetic engineering bacterium CstrxR1 generation ability and is significantly improved.
SEQUENCELISTING
<110>Nanjing Forestry University
<120>one strain Filamentous fungi engineering bacteria CstrxR1 and construction method thereof and application
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gtgaacggcctgttctacgccgttggtcacgagcccgcgaccggcctcgtccgcggccag840
ctgcagacagacaccgatggctacatcatcaccgtccctggcacgacccagacgagcgtc900
aagggtgtcttcgcggcaggtgacgtgcaggacaagaggtaccgtcaggcgatcaccagt960
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MetAlaProLeuThrAsnGlyAlaAsnGlyGluValGlnLysThrGly
151015
GluGlnSerSerLysLeuHisSerLysValValIleIleGlySerGly
202530
ProAlaGlyHisThrAlaAlaIleTyrLeuAlaArgAlaAsnLeuAsn
354045
ProValLeuPheGluGlyPheMetAlaAsnGlyPheAlaAlaGlyGly
505560
GlnLeuThrThrThrThrGluIleGluAsnPheProGlyPheProSer
65707580
GlyIleLeuGlyProGluLeuMetAspArgPheArgAlaGlnSerLeu
859095
ArgPheGlyThrAspIleIleThrGluThrIleSerLysValAspLeu
100105110
SerGlnArgProPheArgTyrTrpArgGluGlyGlnGluThrGluGlu
115120125
ProGluThrAlaAspThrLeuIleIleAlaThrGlyAlaSerAlaLys
130135140
ArgLeuGlyLeuLysGlyGluGluAlaTyrTrpGlnSerGlyIleSer
145150155160
AlaCysAlaValCysAspGlyAlaValProIlePheArgAsnLysPro
165170175
LeuAlaValIleGlyGlyGlyAspSerAlaAlaGluGluAlaThrTyr
180185190
LeuThrLysTyrGlySerHisValTyrValLeuValArgArgGlyGlu
195200205
LeuArgAlaSerLysIleMetAlaLysArgLeuMetAsnHisProLys
210215220
ValThrIleLeuTrpAsnThrValAlaValGluCysGlnGlyAspGly
225230235240
AspLeuLeuAsnAsnLeuArgIleLysAsnValLeuThrGlyGluGlu
245250255
GlnAspLeuGlnValAsnGlyLeuPheTyrAlaValGlyHisGluPro
260265270
AlaThrGlyLeuValArgGlyGlnLeuGlnThrAspThrAspGlyTyr
275280285
IleIleThrValProGlyThrThrGlnThrSerValLysGlyValPhe
290295300
AlaAlaGlyAspValGlnAspLysArgTyrArgGlnAlaIleThrSer
305310315320
AlaGlySerGlyCysMetAlaAlaLeuGluAlaGluArgLeuIleSer
325330335
GluGluGluGluGlyIleGluAsp
340
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Claims (5)

1. a strain Filamentous fungi engineering bacteria CstrxR1, specific name is Filamentous fungi TrichodermareeseiCstrxR1, it is preserved in China typical culture collection center, preservation date is: on March 1st, 2016, deposit number is CCTCCNO:M2016078, and preservation address is Hubei China Wuhan Wuhan University.
2. Filamentous fungi engineering bacteria CstrxR1 according to claim 1, it is characterised in that clone has the thioredoxin reductase gene order coming from worm plan wax bacterium in described Filamentous fungi engineering bacteria CstrxR1, and this gene order is such as shown in SEQIDNO.1.
3. build the method for Filamentous fungi engineering bacteria CstrxR1 described in claim 2, it is characterized in that: by sequence, the such as DNA fragmentation shown in SEQIDNO.1 imports Filamentous fungi genome by plasmid pAg1-CstrxR1, described plasmid pAg1-CstrxR1 loads Filamentous fungi cellobiohydrolase I promoter sequence Pcbh1, the sequence such as DNA fragmentation shown in SEQIDNO.1 and Filamentous fungi cellobiohydrolase I terminator sequence Tcbh1 in KpnI and the ApaI site of plasmid pAg1-H3 order.
4. the method building Filamentous fungi engineering bacteria CstrxR1 according to claim 3, it is characterised in that: described Filamentous fungi is Filamentous fungi D-86271.
5. the application in cellulase-producing of the Filamentous fungi engineering bacteria CstrxR1 described in claim 1.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1712534A (en) * 2004-06-25 2005-12-28 私立逢甲大学 Nucleic acid structure and expression carrier for enhancing recombinant protein production and mass-production of recombinant protein
CN101942478A (en) * 2010-08-31 2011-01-12 上海交通大学 Foreign protein soluble expression plasmid, preparation method thereof and application method thereof
CN102533835A (en) * 2010-08-31 2012-07-04 上海交通大学 Plasmid for heterologous protein solubility expression and preparation and application method thereof
CN102719473A (en) * 2012-06-28 2012-10-10 中国科学院微生物研究所 Acremonium-chrysogenum engineering bacterium and construction method thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1712534A (en) * 2004-06-25 2005-12-28 私立逢甲大学 Nucleic acid structure and expression carrier for enhancing recombinant protein production and mass-production of recombinant protein
CN101942478A (en) * 2010-08-31 2011-01-12 上海交通大学 Foreign protein soluble expression plasmid, preparation method thereof and application method thereof
CN102533835A (en) * 2010-08-31 2012-07-04 上海交通大学 Plasmid for heterologous protein solubility expression and preparation and application method thereof
CN102719473A (en) * 2012-06-28 2012-10-10 中国科学院微生物研究所 Acremonium-chrysogenum engineering bacterium and construction method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LIN,C.-T等: "登录号：ABL61265.1", 《GENBANK》 *
REESEIGUOKUN WANG等: "Mitochondria thioredoxin’s backup role in oxidative stress resistance resistancein Trichoderma reesei", 《MICROBIOLOGICAL RESEARCH》 *
安乃莉等: "应用硫氧还蛋白促进外源蛋白在大肠杆菌的可溶性表达", 《病毒学报》 *

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